Plant with modified ribulose-1,5-bisphosphate carboxylase / oxygenase

Abstract

For the purpose of producing plants having improved growth capacity (plant individual size, growth rate, etc.), the present invention addresses the problem of producing rubisco that improves the plant growth capacity. Specifically, the present invention is a method for producing a plant having improved growth capacity. The method includes introducing, into the rbcL gene sequence of the plant, a base substitution that mutates the 309th methionine of the large subunit (RbcL) of ribulose-1,5-bisphosphate carboxylase / oxygenase (rubisco) of the plant to a different amino acid and / or a base substitution that mutates the 397th aspartic acid of the RbcL of the plant to a different amino acid.

Description

Ribulose-1,5-bisphosphate carboxylase / oxygenase modified plants

[0001] This invention relates to the modification of plant ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco).

[0002] As climate change accelerates, rising atmospheric CO2 concentrations are further exacerbating the global temperature increase since the Industrial Revolution, exacerbating the food crisis. Meanwhile, demand for food is increasing year by year, and to meet this demand, it is necessary to increase the production of crops, which are the foundation of the food supply. Furthermore, plants are highly sensitive to climate change and have a significant impact on regulating atmospheric CO2 concentrations. Ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) (hereinafter also referred to as "Rubisco"), which plays a crucial role in photosynthesis, has low catalytic efficiency, and under physiological conditions, its catalytic reaction is the rate-limiting factor in CO2 fixation during photosynthesis. Therefore, there is hope for the creation of plants that possess Rubisco, which enables a high carboxylation rate, in order to increase crop yields and lower atmospheric CO2 levels.

[0003] Modification of Rubisco in plants has primarily been carried out using tobacco (Nicotiana tabacum), for which methods of chloroplast transformation have been established. Rubisco consists of 16 subunits: eight large subunits (RbcL) encoded by rbcL in the chloroplast genome and eight small subunits (RbcS) encoded by rbcS in the nuclear genome. Attempts have been made to identify novel Rubisco variants with high catalytic rates from naturally occurring plants and algae, or to create hybrid Rubisco by replacing endogenous plant Rubisco with Rubisco from other plant species (Non-Patent Documents 1 to 10). However, creating plants with desired capabilities using such high-performance Rubisco has been difficult due to the poor efficiency of subunit association and the difficulty in dissolving ectopically expressed Rubisco.

[0004] In recent years, several amino acid residues in RbcL that are important for improving the carboxylation rate of Rubisco have been identified through evolutionary approaches to Rubisco using selection by Rubisco-dependent Escherichia coli (Non-Patent Literature 11), and through research on the molecular evolution from C3 to C4 plants of the genus Flavelia (Non-Patent Literature 12). However, it remains unclear whether Rubisco with artificially substituted amino acid residues functions in plants and produces beneficial effects on the growth of individual plants. As long as conventional chloroplast transformation methods are used, the number of plants that can be produced is limited, and the resulting chloroplast-transformed plants are treated as genetically modified plants, making regulatory hurdles high. Furthermore, it is difficult to introduce only specific base substitutions (amino acid substitutions) into the chloroplast genome, making it extremely difficult to produce plants with Rubisco that exhibit the desired performance.

[0005] Sharwood et al., Current Opinion in Plant Biology 31, 135-142 2016. Conlan and Whitney, Nature Plants 4, 12-13 2018. Flamholz et al., Biochemistry 58, 3365-3376 2019. Davidi D et al., The EMBO Journal 39, e104081 2020. Matsumura et al., Molecular Plant 13, 1570-1581 2020. Lin et al., Nature 513, 547-550 2014. Occhialini et al., The Plant Journal 85, 148-160 2016. Long et al., Nature Communications 9, 3570 2018. Gunn et al., Proceedings of the National Academy of Sciences 117, 25890-25896 2020. Orr et al., Plant Physiology 182, 807-818 2020. Wilson et al., Journal of Biological Chemistry 293, 18-27 2018. Whitney et al., Proceedings of the National Academy of Sciences 108, 14688-14693 2011. Nakazato et al., Nature Plants 7, 906-913 2021. Mok et al., Nature Plants 8, 1378-1384 2022. Mok et al., Nat. Biotechnol. 40, 1378-1387 2022

[0006] In view of the above circumstances, the present invention aims to produce a Rubisco that improves the growth capacity of plants, with the objective of creating plants with improved growth capacity (e.g., size of individual plants, growth rate, etc.). In particular, considering the current and future increase in atmospheric CO2 concentration, the objective is to produce a Rubisco that improves plant growth capacity even at high CO2 concentrations.

[0007] The efficiency of photosynthesis is a major factor influencing plant growth. Improving the function of Rubisco, the rate-limiting enzyme in photosynthesis, is considered one effective way to improve photosynthesis efficiency. The inventors used ptpTALECD (Non-Patent Literature 13 and Non-Patent Literature 14) and ptpTALECD_v2mod (which has DddA11, a highly active cytidine deaminase, instead of DddA; Non-Patent Literature 15), single-nucleotide substitutions to rbcL (the gene encoding the Rubisco large subunit) in Arabidopsis thaliana, and created several mutant Arabidopsis thaliana with amino acid substitutions in RbcL (the Rubisco large subunit). Among the Rubisco mutants created, those with the amino acid substitution Met-309-Ile (M309I) or Asp-397-Asn (D397N) in RbcL showed increased catalytic turnover rate (kcat) and no adverse effects on other Rubisco functions. Plants possessing these Rubisco mutants were found to be in the current atmospheric CO2 concentration (389 μmol mol). -1 ) Not only below, but also the predicted atmospheric CO2 concentration in the future (549 μmol mol -1 Under these conditions, the rate of photosynthesis was also increased. The catalytic capacity of Rubisco composed of RbcL artificially substituted with M309I or D397N is improved, and the growth capacity of plants possessing said Rubisco is also improved, which has been revealed for the first time by the present inventors. The present invention was completed based on the above findings.

[0008] In other words, the present invention is as follows (1) to (13): (1) A method for producing a plant with improved growth ability, comprising introducing into the rbcL gene sequence of the plant a base substitution that changes the 309th methionine of the large subunit (RbcL) of ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) of the plant to another amino acid, and / or a base substitution that changes the 397th aspartic acid of the RbcL of the plant to another amino acid. (2) The method for producing the plant according to (1), comprising introducing into the rbcL gene sequence of the plant a base substitution that changes the 309th methionine to isoleucine. (3) The method for producing the plant according to (2), wherein the base substitution that changes the 309th methionine to isoleucine is a substitution that converts the 927th G:C pair in the base sequence of the rbcL gene to an A:T pair. (4) The method for producing a plant according to (1), comprising introducing a base substitution that causes the 397th aspartic acid to be replaced with asparagine into the rbcL gene sequence of the plant. (5) The method for producing a plant according to (4), wherein the base substitution that causes the 397th aspartic acid to be replaced with asparagine is a substitution that converts the 1189th G:C pair in the base sequence of the rbcL gene to an A:T pair. (6) The method for improving the growth capacity of a plant, comprising introducing a base substitution that causes the 309th methionine in the RbcL of the plant to be replaced with another amino acid, and / or a base substitution that causes the 397th aspartic acid in the RbcL of the plant to be replaced with another amino acid, into the rbcL gene sequence of the plant. (7) A method for enhancing the catalytic activity of Rubisco, comprising the steps of mutating the 309th methionine of the RbcL of Rubisco to another amino acid, and / or mutating the 397th aspartic acid of the RbcL of Rubisco to another amino acid. (8) The method according to (7), comprising the step of introducing a base mutation that causes an M309I substitution and / or a D397N substitution into the DNA sequence encoding the RbcL. (9) A plant produced by the manufacturing method described in any of (1) to (5) above, and a descendant of said plant. (10) Seeds obtained from the plant described in (9) above or from a descendant of said plant.(11) A plastid genome into which a base substitution is introduced that causes the 309th methionine of RbcL to be replaced with another amino acid, and / or a base substitution is introduced that causes the 397th aspartic acid of RbcL to be replaced with another amino acid, a plastid having said plastid genome, a plant cell having said plastid genome, a seed containing said plant cell, or a plant containing said plant cell. (12) A plastid genome as described in (11) above, a plastid having said plastid genome, a plant cell having said plastid genome, a seed containing said plant cell, or a plant containing said plant cell, into which a base substitution is introduced that causes the 309th methionine to be replaced with isoleucine. (13) A plastid genome as described in (11) above, a plastid having said plastid genome, a plant cell having said plastid genome, a seed containing said plant cell, or a plant containing said plant cell, into which a base substitution is introduced that causes the 397th aspartic acid to be replaced with asparagine. In this specification, the symbol "~" indicates a numerical range including the values ​​to the left and right of it.

[0009] According to the present invention, it is possible to produce Rubisco with improved catalytic activity. Furthermore, plants possessing this Rubisco have higher growth capacity than the wild type, not only under current atmospheric CO2 concentrations but also under higher CO2 concentrations. Therefore, the present invention will enable increased production of plants, especially food crops, from the present into the future.

[0010] Figure 1 shows the atmospheric CO2 concentration (381 ± 14 μmol mol). -1 ) under conditions and high CO2 concentration (549±23 μmol mol -1The following shows the results of investigating the growth status of plants 48 days after sowing under the following conditions. a and b are representative images of plants cultivated under atmospheric CO2 concentration conditions (a) and high CO2 concentration conditions (b). c to e show the total leaf area (c), total dry weight of shoots (d), and total dry weight of roots for plants cultivated under atmospheric CO2 concentration conditions. f and g show the total leaf area (f) and total dry weight of shoots (g) for plants cultivated under high CO2 concentration conditions. Values ​​are shown as mean ± standard error. Each data point is indicated by a black dot. ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n=6. Figure 2 shows the results of enzyme characterization analysis of each Rubisco mutant, and the measurement results of Rubisco content and chlorophyll content in plant individuals containing the Rubisco mutant. a-d show the kcat (a), Kc (b), kcat / Kc (c), and Sc / o (d) of each mutant Rubisco. e shows the Rubisco content in mutant plant individuals of different strains. f shows the chlorophyll content in mutant plant individuals of different strains. Values ​​are shown as mean ± standard error. Each data point is indicated by a black dot. ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n=6. Figure 3 shows the measurement results of photosynthetic parameters for each mutant plant. a shows the results of measuring the CO2 fixation rate of different mutant strains under various intracellular CO2 concentration conditions, among the gas exchange parameters. b shows the results of measuring the electron transport rate of PSII of different mutant strains under various intracellular CO2 concentration conditions, among the gas exchange parameters. c-f are 400 μmol mol -1 The following shows the results of measuring gas exchange parameters (CO2 fixation rate (c), stomatal conductance (d), intracellular CO2 concentration (e), and water use efficiency (A / gs) (f)) of different mutant lines under CO2 concentration conditions. g and h are 400 μmol mol -1This figure shows the results of measuring the electron transport rate (g) and NPQ (h) of PSII, which are photosynthetic fluorescence parameters, under CO2 conditions. Values ​​are shown as mean ± standard error. Each data point is indicated by a black dot. ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n=6. Figure 4 summarizes the results of T1 generation base editing. a is a schematic diagram showing the binding between the base editing enzyme and its target sequence. Platinum TALE is the DNA-binding domain of platinum TALEN, CD is cytidine deaminase, and UGI is uracil glycosylase inhibitor. b-g show the base editing efficiency of the T1 generation. The table in the upper panel shows which bases in the target window were edited, to what frequency, and how many plant individuals underwent base editing. The genotype was determined by Sanger sequencing, and representative Sanger sequence waveforms are shown in the lower panel. The constructed constructs are rbcL_M309I-3_DddA11 (b), rbcL2_1397NC (c), rbcL2-3_1333NC (d), rbcL1_1397CN (e), rbcL3-2_1397CN (or NC) (f), and rbcL4_1397CN (g). Homo indicates homoplasmic editing, and h / c indicates heteroplasmic or chimeric editing.

[0011] The following describes embodiments for carrying out the present invention. When referring to "this embodiment," unless otherwise specified, it refers to all embodiments described herein. The first embodiment is a method for producing plants with improved growth ability, comprising introducing a base substitution into the rbcL gene sequence of the plant, which involves changing the 309th methionine of the ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) macrosubunit (RbcL) of the plant to another amino acid, and / or changing the 397th aspartic acid of the Rubisco macrosubunit of the plant to another amino acid (hereinafter also referred to as "the production method according to this embodiment"). Regarding the amino acid sequence of the Rubisco macrosubunit (hereinafter also referred to as "RbcL") in this embodiment, for example, the amino acid sequence represented by the following SEQ ID NO: 1 can be used as an example of the amino acid sequence of the Rubisco macrosubunit of Arabidopsis thaliana; SEQ ID NO: 1 (RbcL amino acid sequence) MSPQTETKASVGFKAGVKEYKLTYYTPEYETKDTDILAAFRVTPQPGVPPEEAGAAVAAESSTGTWTTVWTDGLTSLDRYKGRCYHIEPVPGEETQFIAYVAYPLDLFEEGSVTNMFTS IVGNVFGFKALAALRLEDLRIPPAYTKTFQGPPHGIQVERDKLNKYGRPLLGCTIKPKLGLSAKNYGRAVYECLRGGLDFTKDDENVNSQPFMRWRDRFLFCAEAIYKSQAETGEIKGHY LNATAGTCEEMIKRAVFARELGVPIVMHDYLTGGFTANTSLSHYCRDNGLLLHIHRAMHAVIDRQKNHGMHFRVLAKALRLSGGDHIHAGTVVGKLEGDRESTLGFVDLLRDDYVEKDRS RGIFFTQDWVSLPGVLPVASGGIHVWHMPALTEIFGDDSVLQFGGGTLGHPWGNAPGAVANRVALEACVQARNEGRDLAVEGNEIIREACKWSPELAAACEVWKEITFNFPTIDKLDGQE

[0012] Since rbcL is reported to be among the top 1% of genes with the slowest evolutionary rate (Bouvier et al., Proceedings of the National Academy of Sciences 121, 2024, e2321050121), its amino acid sequence is considered to be widely conserved among organisms. In this specification, "RbcL" refers to a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, as well as a protein consisting of an amino acid sequence in which, preferably, about 1 to 60, 1 to 50, more preferably 1 to 40, 1 to 30, preferably 1 to 20, even more preferably 1 to 10, and even more preferably 1 to 5 amino acids are deleted, substituted, inserted, or added from the amino acid sequence represented by SEQ ID NO: 1. Alternatively, in this specification, "RbcL" is a protein comprising an amino acid sequence having about 60% or more, preferably about 70% or more, more preferably about 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, and most preferably about 99% amino acid identity with the amino acid sequence represented by Sequence ID No. 1.

[0013] Furthermore, the 309th methionine and 397th aspartic acid of RbcL are known to be conserved in many plants (see Table 3 in the Examples). In this specification, the "309th methionine of the Rubisco macrosubunit" in plants other than Arabidopsis thaliana refers to the methionine on the amino acid sequence of the Rubisco macrosubunit of the other plant that corresponds to the 309th position on the amino acid sequence of Sequence ID No. 1 when the amino acid sequence of Sequence ID No. 1 and the amino acid sequence of the Rubisco macrosubunit of the other plant are aligned to maximize the degree of agreement between the two. Similarly, the "397th aspartic acid of the Rubisco macrosubunit" in plants other than Arabidopsis thaliana refers to the aspartic acid on the amino acid sequence of the Rubisco macrosubunit of the other plant that corresponds to the 397th position on the amino acid sequence of Sequence ID No. 1 when the amino acid sequence of Sequence ID No. 1 and the amino acid sequence of the Rubisco macrosubunit of the other plant are aligned to maximize the degree of agreement between the two. Hereafter, "methionine at position 309 of the Rubisco macrosubunit" will also be referred to simply as "methionine at position 309" or "M309," and "aspartic acid at position 397 of the Rubisco macrosubunit" will also be referred to simply as "aspartic acid at position 397" or "D397."

[0014] The mutation at M309 in RbcL may be a mutation to any amino acid, but is preferably a mutation to isoleucine (M309I). Similarly, the mutation at D397 in RbcL may be a mutation to any amino acid, but is preferably a mutation to asparagine (D397N). The mutation at M309 in RbcL to isoleucine (also referred to as "M309I") or the mutation at D397 to asparagine ("D397N") can be introduced by a method readily selectable by those skilled in the art. For example, such a method involves using ptpTALECD (see Non-Patent Documents 13 and 14, etc.) to replace a C:G (G:C) pair with a T:A (A:T) pair in the region encoding M309 or D397 in the rbcL gene sequence. More specifically, to produce the M309I amino acid mutation, the 927th G:C pair in the rbcL gene sequence (the sequence encoding RbcL; for example, the RbcL encoding sequence of Arabidopsis thaliana is shown in SEQ ID NO: 2) may be converted to an A:T pair, and to produce the D397N amino acid mutation, the 1189th G:C pair in the rbcL gene sequence (the sequence encoding RbcL; for example, the RbcL encoding sequence of Arabidopsis thaliana is shown in SEQ ID NO: 2) may be converted to an A:T pair. It is desirable that these single nucleotide substitutions be introduced homoplasmically into the plastid genome.

[0015]

[0016] The production of plants with mutations at D309 and M397 of RbcL can be carried out by methods chosen by those skilled in the art, but the nucleotide substitutions that cause the amino acid mutations in the rbcL gene may also be introduced into the plastid genome using the aforementioned ptpTALECD (Non-Patent Documents 13 and 14) and ptpTALECD_v2mod (Non-Patent Document 15). Specifically, for example, a binary vector encoding ptpTALECD (a single-nucleotide substitution editing enzyme gene) is introduced into the nuclear genome of a plant using the floral dip method (Clough et al., Plant J. 16, 735-743 1998), and T1 seeds are collected from the plant. From the collected T1 seeds, seeds into which the single-nucleotide substitution editing enzyme gene has been introduced are selected using selection markers or the like as indicators. The selected T1 seeds are sown, and individuals with the target base substitution are identified by Sanger sequencing or the like. From T2 seeds collected from plants grown from identified individuals, seeds in which the single-nucleotide substitution editing enzyme gene has been removed from the nuclear genome are selected using the absence of the selection marker as an indicator. The T2 seeds obtained in this way have the desired single-nucleotide substitution in the plastid genome and do not have the single-nucleotide substitution editing enzyme gene, so further single-nucleotide substitutions (unintended substitutions) are not induced in the plastid genome. See the examples for details. Desired plants can be grown from these T2 seeds.

[0017] The manufacturing method according to this embodiment is a method for producing plants with improved growth ability by starting with a plant in which the amino acid corresponding to the 309th position in the amino acid sequence of RbcL is methionine, or a plant in which the amino acid corresponding to the 397th position in the amino acid sequence of RbcL is aspartic acid, and introducing a base substitution into the rbcL gene sequence. Here, the starting plant can be, for example, many C3 plants (plants that perform photosynthetic carbon assimilation only through the Calvin cycle, and whose first photosynthetic fixation product is a three-carbon compound. For example, crops such as rice, wheat, soybeans, rapeseed, and spinach are included).

[0018] Introducing the M309 mutation (e.g., M309I) and / or the D397 mutation (e.g., D397N) into RbcL increases the catalytic velocity (kcat) of RbcL, and the CO2 fixation rate and electron transport rate increase in the photosynthetic reaction of plants containing RbcL. As a result, the growth capacity of plants with these mutations improved compared to plants without the mutation. Since this increase in the catalytic velocity of RbcL and the increase in the photosynthetic reaction rate of plants containing RbcL are observed not only under current atmospheric CO2 concentrations but also under higher CO2 concentrations predicted for the future, it is considered that plants produced by the manufacturing method according to this embodiment will continue to exhibit high growth capacity in the future.

[0019] In this embodiment, whether or not "growth capacity" has improved may be evaluated using indicators of the external or physiological characteristics of the plant individual that are affected by the increase in the photosynthetic reaction rate, and is not particularly limited. For example, the growth rate of the plant individual, including the roots, leaves, or stems, the dry weight of the roots or shoots, the total leaf area, the number of flowers, fruits, or seeds may be used as indicators, and if these indicators increase, it can be determined that the plant's "growth capacity" has improved. However, the evaluation of "growth capacity" may also be performed using indicators other than those exemplified herein, which can be easily set by those skilled in the art.

[0020] The second embodiment is a plant (adult plant) produced by the manufacturing method according to the first embodiment (i.e., the manufacturing method according to this embodiment), its offspring plants, and seeds obtained from said plant and its offspring plants. More specifically, the second embodiment is a plant with improved growth capacity in which a base substitution is introduced into the rbcL gene sequence, which replaces the 309th methionine in the large subunit (RbcL) of ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) with another amino acid, and / or replaces the 397th aspartic acid in said RbcL with another amino acid, its offspring plants, and seeds obtained from said plant and its offspring plants. In other words, the second embodiment includes not only the plant (T0 generation) produced by the manufacturing method according to the first embodiment and the seeds obtained from said plant (T1 seeds), but also the offspring plants of said plant (T1 generation and later plants) and seeds obtained from said offspring plants (T2 seeds and later seeds). For further details regarding the second embodiment, please refer to the description of the first embodiment.

[0021] The third embodiment is a method for improving the growth capacity of a plant, comprising introducing a base substitution into the rbcL gene sequence of the plant, which replaces the 309th methionine in the RbcL of the plant with another amino acid, and / or replaces the 397th aspartic acid in the RbcL of the plant with another amino acid. The method according to the third embodiment is a method for improving the growth capacity of a plant by introducing a base substitution into the rbcL gene sequence of a plant in which the amino acid corresponding to the 309th position in the amino acid sequence of the RbcL is methionine, or a plant in which the amino acid corresponding to the 397th position in the amino acid sequence of the RbcL is aspartic acid. For further details regarding the third embodiment, please refer to the description of the first embodiment.

[0022] A fourth embodiment is a method for enhancing the catalytic activity of Rubisco, comprising the steps of mutating the 309th methionine of the large subunit of Rubisco (RbcL) with another amino acid, and / or mutating the 397th aspartic acid of RbcL with another amino acid. Here, it is preferable that the 309th methionine of RbcL be mutated with isoleucine, and it is preferable that the 397th aspartic acid be mutated with asparagine. Such amino acid substitutions may be performed in vivo or in vitro and can be easily carried out using well-known techniques selectable by those skilled in the art. Briefly speaking, examples include introducing a base mutation that causes an M309I substitution or a D397N substitution into the gene sequence (i.e., rbcL) encoding RbcL (in vivo) or into the DNA sequence (in vitro). As an indicator of the catalytic activity of Rubisco, for example, catalytic rate (kcat) can be used, but is not limited to this.

[0023] A fifth embodiment is a plastid (e.g., chloroplast) genome, a plant cell having said plastid genome, a seed containing said plant cell, or a plant (adult plant) containing said plant cell, in which a base substitution is introduced (artificially introduced) into the rbcL gene sequence, which replaces the 309th methionine of the large subunit (RbcL) of ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) with another amino acid, and / or replaces the 397th aspartic acid of said RbcL with another amino acid.

[0024] In the fifth embodiment, the plant from which the RbcL is derived before the mutation is introduced (i.e., RbcL in which the 309th amino acid is methionine and / or the 397th amino acid is aspartic acid) can be, for example, many plants belonging to C3 plants (see above). The mutation at M309 in RbcL may be a mutation to any amino acid, but is preferably a mutation to isoleucine. Similarly, the mutation at D397 in RbcL may be a mutation to any amino acid, but is preferably a mutation to asparagine. For methods of introducing mutations into RbcL, and for methods of producing plant cells, seeds, and plants (adult plants) having the introduced plastid, please refer to the description of the first embodiment.

[0025] The plants in this embodiment (all of the first to fifth embodiments) are not particularly limited and may include not only edible plants (agricultural crops such as vegetables, grains, and fruit trees) but also ornamental plants (flowering plants, foliage plants, etc.). If we were to list some examples of such plants, they would be, for example, grasses; plants of the genus Poaceae (e.g., rice), the genus Wheat (e.g., wheat), the genus Barley (e.g., barley), and the genus Rye (e.g., rye); plants of the genus Brassica (e.g., tatasai, mustard greens, mustard greens, rapeseed, mizuna, kale, ornamental cabbage, cauliflower, cabbage, Brussels sprouts, broccoli, bok choy, Nozawana, rapeseed, Chinese cabbage, komatsuna, turnip, etc.) ), *Thicus* genus (e.g., *Thicus gracilis*), *Arabidopsis* genus (e.g., *Arabidopsis thaliana*), *Capsella* genus (e.g., *Capsella bursa-pastoris*), *Arugula* genus (e.g., *Arugula serrata*), *Rapeseed* genus (e.g., *Rapeseed*), *Rapeseed* genus (e.g., *Stock*), *Watercress* genus (e.g., *Watercress*), *Radish* genus (e.g., *Radish*, *Radish radish*, etc.), *Wasabi* genus (e.g., *Wasabi*), and *Wasabi* genus (e.g., *Wasabi*). Plants such as horseradish, plants of the Solanaceae family; plants of the Solanum genus (e.g., eggplant, tomato, potato, etc.), Capsicum genus (e.g., chili pepper, bell pepper, paprika, shishito pepper, etc.), Ipomoea genus (e.g., petunia), and Physalis genus (e.g., physalis), plants of the Asteraceae family; plants of the Chrysanthemum genus (e.g., chrysanthemum, garland chrysanthemum, etc.), Helianthus genus (e.g., sunflower, Jerusalem artichoke, etc.), Taraxacum genus (e.g., Japanese dandelion, Japanese dandelion, European dandelion, etc.), and autumn berries. Plants such as those belonging to the genera *Lactobacillus* (e.g., lettuce), *Actula* (e.g., burdock), *Pepper* (e.g., butterbur), *Farfugium* (e.g., leopard plant), and *Smaranthus* (e.g., yacon), belonging to the Convolvulaceae family; plants such as those belonging to the genera *Ipomoea* (e.g., sweet potato, morning glory, bindweed, moonflower, water lily), belonging to the Araceae family; plants such as those belonging to the genera *Colocasia* (e.g., taro, mountain taro, etc.) and *Amorphophallus* (e.g., konjac, mountain konjac, elephant konjac, titan konjac, etc.), belonging to the Fabaceae family;Plants such as those belonging to the genera *Phragmites* (e.g., kidney beans, lima beans), *Glycine* (e.g., soybeans), *Vigna* (e.g., adzuki beans, mung beans, cowpeas), *Vicia* (e.g., broad beans), *Pisces* (e.g., peas), and *Caulerpa* (e.g., peanuts), plants of the Cucurbitaceae family; plants belonging to the genera *Cucumber* (e.g., cucumbers, melons, cantaloupes, white melons, horned melons), and *Cucurbita* (e.g., watermelons) Plants such as the genus Cucurbita (e.g., zucchini, European pumpkin, Japanese pumpkin), the genus Lagenaria (e.g., bottle gourd, gourd), the genus Melon (e.g., winter melon), the genus Momordica (e.g., bitter melon, Chinese snake gourd), the genus Monk Fruit (e.g., Monk Fruit), and the genus Luffa (e.g., loofah), plants of the Amaryllidaceae family; the genus Allium (e.g., onion, chives, scallions, tadpoles) Examples include plants such as onions, garlic, shallots, chives, wild onions, and leeks; plants of the Asparagaceae family; plants of the Asparagaceae family; plants of the Apiaceae family; plants of the Apiaceae family (e.g., water dropwort), plants of the Apiaceae family (e.g., parsley), plants of the Coriander family (e.g., coriander or cilantro), plants of the Angelica genus (e.g., Angelica keiskei), plants of the Carrot genus (e.g., carrots), and plants of the Amaranthaceae family; plants of the Amaranthaceae family (e.g., spinach) and plants of the Swiss chard genus (e.g., table beets, sugar beets), etc.

[0026] All disclosures of referenced documents in this specification are incorporated by reference as a whole. Furthermore, throughout this specification, where the singular words “a,” “an,” and “the” are included, they are to be considered plural as well as singular unless the context clearly indicates otherwise. The present invention will be further described below with reference to examples, which are merely illustrative of embodiments of the present invention and do not limit the scope of the invention.

[0027] 1. Method 1-1. Preparation and Introduction of Rubisco Mutants The binary vector encoding ptpTALECD was assembled according to the previously reported method (Non-Patent Literature 13). To construct the binary vector encoding ptpTALECD_v2mod, in the second step of the assembly, a different entry vector (Addgene ID numbers; 191599-191602 and 191607-191610) was used compared to the entry vector of the Platinum Gate TALEN kit. The sequences to which the TALE array binds are shown in Table 1. These sequences were selected based on the characteristics of the location of the cytosine to be edited (Non-Patent Literature 13).

[0028] The binary vector was introduced into the nuclear genome of Arabidopsis thaliana Col-0 using the floral dip method (Clough et al., Plant J. 16, 735-743 1998). T1 seeds were selected based on the observation of GFP fluorescence derived from the introduced gene (Shimada et al., Plant J. 61, 519-528 2010). T1 leaves were incubated in 50 μL of TE Buffer (100 mM Tris-HCl (pH 9.5), 10 mM EDTA (pH 8.0)) at 98°C for 15 minutes to extract DNA. The target sequence was amplified using KOD One® PCR Master Mix (TOYOBO). The PCR product was purified using FastGene® Gel / PCR Extraction Kit (Nippon Genetics) and sequenced by Sanger sequencing. The primer sequences for PCR are shown in Table 2. Null isolated T2 seeds were selected based on the absence of GFP fluorescence. Genotyping of T2 plants was performed in the same manner as for T1 plants.

[0029] 1-2. Plant Materials and Cultivation Conditions Seeds of Arabidopsis thaliana (L.) were sown in a 1:1 mixture of Metromix 350 (Hyponex) and vermiculite and cultivated in an environmentally controlled growth chamber. The growth chamber was set with a light period and a dark period of 10 hours and 14 hours, respectively, and the photosynthetic photon flux density (PPFD) during the light period was set to 150 μmol photons m -2 s -1 . Also, the temperature and relative humidity were set to be constant at 22°C and 60%, respectively. The CO2 concentration in the chamber was set to the same concentration as the atmospheric concentration (381 ± 20 μmol mol -1 ), a concentration higher than the atmospheric concentration (549 ± 23 μmol mol -1 ), and a concentration lower than the atmospheric concentration (286 ± 14 μmol mol -1 ).

[0030] 1-3. Plant Growth Plants were cultivated under three different CO2 concentration conditions (ambient CO2 concentration, high CO2 concentration, and low CO2 concentration). To examine the growth state at the initial stage of cultivation, the plants were harvested on the 25th day after cultivation (ambient CO2 concentration and high CO2 concentration) and on the 32nd day after cultivation (low CO2 concentration). To examine the growth state at the later stage of cultivation, the plants were harvested on the 48th day after cultivation (under ambient CO2 concentration and high CO2 concentration) and on the 47th day after cultivation (low CO2 concentration). During cultivation, all above-ground tissues were separated for each individual. Also, the leaf area was scanned using a scanner, and the area was calculated using ImageJ software. After scanning the leaves, all above-ground tissues were collected and dried at 60°C for 48 hours to measure the dry weight. The underground tissues were carefully pulled out without damaging the root system and washed with running water. Then, the root tissues were dried at 60°C for 48 hours, and the dry weight was measured.

[0031] 1-4. Analysis of Photosynthetic Reactions under Various CO2 Concentration Conditions: Gas exchange rate and chlorophyll fluorescence were simultaneously measured on fully opened young leaves of 6- to 8-week-old plants grown at atmospheric CO2 concentrations using a portable gas exchange system (LI-6400XT, LI-6400-40 leaf chamber fluorometer, LI-COR). CO2 fixation rate, stomatal conductance, intracellular CO2 concentration, and chlorophyll fluorescence parameters, including electron transport rate and non-photochemical quenching (NPQ) in Optical System II (PSII), were measured per 1000 μmol photons m -2 s -1 PPFD, 60-70% relative humidity, 25°C temperature, and 50 μmol mol -1 ~1500 μmol mol -1 Measurements were taken under the CO2 concentration conditions. To determine the CO2 / O2 specificity (Sc / O) of Rubisco, 125 and 500 μmol photons were measured under the above conditions. -2 s -1 Under two different light intensities, the carbon dioxide compensation point was determined using the gas exchange rate as an indicator. Sc / o was evaluated by the slope of the regression curve of the carbon dioxide compensation point dependence at the measured light intensities (Laisk and Loreto, Plant Physiology 110, 903-912 1996).

[0032] 1-5. Measurement of Rubisco and Chlorophyll ContentsThe same leaf tissue as that used for measuring the gas exchange rate was extracted immediately after the gas exchange measurement, frozen in liquid nitrogen, and stored at -80°C until analysis. The Rubisco content was determined by extracting the bands corresponding to the large and small subunits of Rubisco stained with Coomassie Brilliant Blue R-250 after separation by SDS-PAGE with formamide and using bovine serum albumin (BSA) as a standard (Yamori et al., Plant Cell and Environment 34, 764-777 2011). The chlorophyll content was extracted with 80% v / v acetone and determined by the method reported previously (Porra et al., Biochimica et Biophysica Acta 975, 384-394 1989).

[0033] 1-6. Analysis of Enzyme Characteristics of Rubisco Leaves were homogenized using a cooled pestle and mortar in an extraction buffer (100 mM Bicine-NaOH, 1 mM EDTA, 5 mM MgCl2, 2 mM NaH2PO4, 5 mM DTT, 20 mM ascorbate, 4 mM amino-n-caproic acid, 0.8 mM benzamidine, 0.4% (w / v) bovine serum albumin, and 1% (w / v) polyvinylpolypyrrolidone, pH 8.0). The resulting homogenate was centrifuged at 15,000 × g for 2 minutes at 4°C. Rubisco in the supernatant was activated by pre-incubating it for 15 minutes on ice in 15 mM MgCl2 and 5 mM NaHCO3. The activity of Rubisco was determined by analyzing the incorporation of 14 C] NaHCO3 (specific radioactivity; 37 MBq mmol -1 ) into acid-stable products according to the method reported previously (Non-Patent Document 5) at 25°C. The reaction was carried out with the activated Rubisco in a reaction solution (100 mM Bicine-NaOH, 20 mM MgCl2, 1 mM EDTA, 5 mM DTT, 0.5 mM RuBP, 1.0 W-A units carbonic anhydrase, and 0.5-20 mM NaH 14The reaction was started by adding CO3 (pH 8.2). One minute after the start of the reaction, half a volume of 1 N HCl was added to the reaction mixture to stop the reaction. The acidic reaction mixture was dried to check its acid stability. 14 C was measured by liquid scintillation. The concentration of the catalytic site of Rubisco was determined according to a previous report (Ishikawa et al., Plant Production Science, 12, 345-350 2009), 14 C] 2-carboxyarabinitol bisphosphate (specific radioactivity; 1.85 GBq mmol -1 ) were determined based on the stoichiometric ratios of ). kcat and Kc were determined from six different NaH 14 The activity of Rubisco at CO3 concentrations (0.5-20 mM) was calculated by directly fitting the Michaelis-Menten equation using KaleidaGraph data analysis software (Synergy Software).

[0034] 1-7. Statistical Analysis Significant differences in variability between the mean values ​​of multiple plant lines and the control group were evaluated using Dunnett's test. Statistical analysis was performed using Prism v. 8.0.1 software.

[0035] 2. Results 2-1. Amino Acid Modification of RbcL As candidate amino acids to be modified, we focused on eight amino acid substitutions in the Rubisco large subunit (RbcL) [M309I, L74M, I393M, D397N, A398T, A414T, P415A, A375V]. Of these eight amino acid substitutions, M309I, D397N, and A414T can be performed by base substitution from C:G pair to T:A pair in the rbcL gene (encoded in the chloroplast genome). For the other five amino acid substitutions, we investigated whether substitution with amino acids highly similar to the substituted amino acids (i.e., 74M, 393M, 398T, 415A, 375V) was possible by base substitution from C:G pair to T:A pair, and decided to try introducing L74F and P415S. L74, M309, D397, A414, and P415 are conserved in many crop species (see Table 3), and if the usefulness of these amino acid substitutions is demonstrated in experiments using Arabidopsis thaliana, it is expected that these amino acid substitutions can be rapidly introduced into various crop species using genome editing technology. To introduce five amino acid substitutions (i.e., M309I, D397N, A414T, L74F, P415S), binary vectors encoding ptpTALECD or ptpTALECD_v2mod were constructed and introduced into the nuclear genome of Arabidopsis thaliana Col-0 using the floral dip method. ptpTALECD_v2mod contains DddA11 (Non-Patent Literature 14), a cytidine deaminase with high base substitution activity. Sequences containing the target bases were amplified by PCR in the T1 generation of transformants, and the base sequences of the purified PCR products were determined by Sanger sequencing. As a result, individuals were obtained in which all five target amino acid substitutions were introduced homoplasmically (i.e., all of the corresponding base substitutions in the plastid genome, which contains hundreds to thousands of copies in the cell) (Figures 4b-g).

[0036] Since many T1 individuals had multiple base substitutions, we investigated whether individuals with only target base substitutions could be obtained in the next generation (T2 generation). As a result, we were able to obtain individuals in which the base substitutions resulting in the amino acid substitutions L74F, M309I, D397N, and A414T were homoplasmically introduced, and no other non-synonymous substitutions were present. Furthermore, since no GFP fluorescence (expressed from a binary vector) was observed from the seeds of these T2 individuals, it was judged that they were likely to be T2 individuals of the nullse-gligant type. Since no additional mutations were introduced in these individuals and their offspring, it is thought that only the already introduced base substitutions affect the phenotype. In total, including by-products (D72N, R312C, and P415L), we were able to obtain seven types of nullse-gligant mutants. Subsequent phenotypic analyses were performed using the T3 or T4 generations of these offspring. Hereafter, the obtained mutants will be referred to by the abbreviations shown in Table 3.

[0037] 2-2. Comparison of growth of various mutants under atmospheric CO2 concentration, high CO2 concentration, and low CO2 concentration conditions. Various Arabidopsis thaliana mutant lines with a single nucleotide substitution introduced in rbcL were subjected to 381 μmol mol -1 Atmospheric CO2 concentration: 549 μmol / mol -1 The plants were cultivated at higher CO2 concentrations (Figures 1a and 1b). At 48 days post-sowing, the M309I-substituted plants and D397N-substituted plants had higher total leaf area and total dry shoot weight compared to the control plants, under both atmospheric CO2 concentration and higher CO2 concentration conditions (Figure 1). In contrast, at 28 days post-sowing, no significant difference was observed between the control group and the M309I-substituted plants in either total leaf area or total dry shoot weight. Furthermore, all plant lines other than the M309I-substituted plants and D397N-substituted plants had lower total leaf area and dry shoot weight compared to the control plants, regardless of CO2 concentration conditions or the timing of post-sowing analysis (Figure 1).

[0038] 2-3. Enzyme Characterization of Rubisco in Various Plant Lineages, and Measurement of Rubisco and Chlorophyll Content The M309I mutant showed a higher catalytic rate (kcat) compared to the control (Figure 2a). The Michaelis-Menten constant (Kc) for CO2 in the M309I mutant was also higher than that of the control. However, the catalytic efficiency of carboxylation (kcat / Kc) and the efficiency of carboxylation relative to oxygenation (Sc / o) in the M309I mutant were lower than those of the control (Figures 2b-d). Furthermore, the D397N mutant showed a higher kcat compared to the control (Figure 2a), and its Sc / o was lower than that of the control (Figure 2d). The Kc and kcat / Kc of the D397N mutant were comparable to those of the control (Figures 2b and c). In contrast, the kcat and kcat / Kc of all other mutants were lower than those of the control (Figures 2a and c). Furthermore, Kc levels in all other mutants were similar to or higher than the control, and Sc / o levels were similar to or lower (Figures 2b and 2d). Regarding Rubisco content, the Rubisco levels in the D72N, M309I, D397N, and A414T mutant plants were similar to the control, but the Rubisco levels in the other mutant plants were lower than the control (Figure 2e). Regarding chlorophyll content, the chlorophyll levels in the D72N, L74F, M309I, D397N, and A414T mutant plants were similar to the control, but the chlorophyll levels in the other mutant plants were lower than the control (Figure 2f).

[0039] 2-4. Analysis of Photosynthetic Reactions in Mutant Plants under Different CO2 Concentration Conditions To investigate the photosynthetic capacity of mutant plants, photosynthetic parameters (CO2 fixation rate; Figure 3a, electron transport rate; Figure 3b) were measured under various CO2 concentration conditions. 400 μmol mol -1Under the specified CO2 concentration conditions, the rbcL M309I and D397N mutants showed significantly higher CO2 fixation and electron transport rates compared to the control, and tended to have lower NPQ (Figure 3c, g, h). In contrast, all other mutants showed lower CO2 fixation and electron transport rates compared to the control (Figure 3c, g). Furthermore, stomatal conductance was equivalent to that of the control in all mutants. Therefore, the intrinsic water-use efficiency (WUEi) (A / gs), which is the CO2 fixation rate divided by stomatal conductance, was higher in the M309I and D397N mutants compared to the control (Figure 3f). These results indicate that the M309I and D397N mutants increase water-use efficiency by improving Rubisco function and mitigating limitations in photosynthesis. In contrast, at 1200 μmol mol -1 Under the specified CO2 concentration conditions, the CO2 fixation rate and electron transport rate were similar in all mutants except R312C and P415L. Furthermore, no significant differences were observed in stomatal conductance, intracellular CO2 concentration, WUEi, and NPQ across all mutants.

[0040] This invention makes it possible to produce plants with superior growth capabilities. Therefore, this invention is expected to be used not only in the agricultural field but also in fields such as food and biofuel production.

Claims

1. A method for producing a plant with improved growth ability, comprising introducing a base substitution into the rbcL gene sequence of the plant, which involves changing the 309th methionine of the large subunit (RbcL) of ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) of the plant to another amino acid, and / or changing the 397th aspartic acid of the RbcL of the plant to another amino acid.

2. The method for producing the plant according to claim 1, comprising introducing a base substitution that changes the 309th methionine to isoleucine into the rbcL gene sequence of the plant.

3. The manufacturing method according to claim 2, wherein the base substitution that changes the 309th methionine to isoleucine is a substitution that converts the 927th G:C pair in the base sequence of the rbcL gene to an A:T pair.

4. The method for producing the plant according to claim 1, comprising introducing a base substitution that changes the 397th aspartic acid to asparagine into the rbcL gene sequence of the plant.

5. The manufacturing method according to claim 4, wherein the base substitution that changes the 397th aspartic acid to asparagine is a substitution that converts the 1189th G:C pair in the base sequence of the rbcL gene to an A:T pair.

6. A method for improving the growth capacity of a plant, comprising introducing a base substitution into the rbcL gene sequence of the plant, which causes the 309th methionine in the RbcL of the plant to be replaced with another amino acid, and / or a base substitution causing the 397th aspartic acid in the RbcL of the plant to be replaced with another amino acid.

7. A method for enhancing the catalytic activity of Rubisco, comprising the steps of mutating the 309th methionine in the RbcL of Rubisco with another amino acid, and / or mutating the 397th aspartic acid in the RbcL of Rubisco with another amino acid.

8. The method according to claim 7, comprising the step of introducing a base mutation that causes an M309I substitution and / or a D397N substitution into the DNA sequence encoding the RbcL.

9. A plant produced by the manufacturing method described in any one of claims 1 to 5, and a descendant of said plant.

10. Seeds obtained from the plant described in claim 9 or from the offspring of said plant.

11. A plastid genome into which a base substitution is introduced that replaces the 309th methionine of RbcL with another amino acid, and / or a base substitution that replaces the 397th aspartic acid of RbcL with another amino acid; a plastid having said plastid genome; a plant cell having said plastid genome; a seed containing said plant cell; or a plant containing said plant cell.

12. A plastid genome according to claim 11, wherein a base substitution is introduced to change the 309th methionine to isoleucine; a plastid having the plastid genome; a plant cell having the plastid genome; a seed containing the plant cell; or a plant containing the plant cell.

13. A plastid genome according to claim 11, wherein a base substitution is introduced to change the 397th aspartic acid to asparagine; a plastid having the plastid genome; a plant cell having the plastid genome; a seed containing the plant cell; or a plant containing the plant cell.

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